The inactivation of the prototype NF-kappaB inhibitor, IkappaBalpha, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation and proteasome-mediated degradation (Ub-Pr). We have identified valosin-containing protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, that co- precipitated with IkappaBalpha immune complexes. The ubiquitinated IkappaBalpha conjugates readily associated with VCP both in vivo and in vitro, and this complex was dissociated from NF-kappaB. Analyses of IkappaBalpha mutants indicated that phosphorylation and ubiquitination were critical for VCP binding which was required for degradation, and the N-terminal domain of IkappaBalpha was required in all three reactions. Density gradient centrifugation analysis showed that complexes containing physically associated VCP and ubiquitinated IkappaBalpha sedimented as 650 kDa molecules with a coefficient of 19S, while the unmodified IkappaBalpha sedimented in 4.5S fractions deficient in VCP. VCP antibodies depleted proteasome activity while immunopurified VCP complexes were sufficient to perform proteasome-like activity, further supporting that VCP is functionally involved in Ub-Pr mediated degradation of IkappaBalpha. VCP co-migrated with subunit 2 (S2) of the 26S proteasome on two dimensional (2D) gels, and was co-purified with S2 in a highly purified proteasome preparation. We propose a model for IkappaBalpha inactivation in which ubiquitinated IkappaBalpha conjugates become physically associated with VCP which displaces the NF-kappaB dimer, thus releasing the dimer for translocation into the nucleus. The ubiquitinated IkappaBalpha is then chaperoned to the 26S proteasome for degradation.